Plasmodium vivax blood stage antigens

ABSTRACT

This invention is directed to novel species-specific P. vivax malarial peptide antigens which are proteins or fragments of proteins secreted into the plasma of a susceptible mammalian host after infection, and to monoclonal or polyclonal antibodies directed against those antigens. The peptide antigens, monoclonal antibodies, and/or polyclonal antibodies are utilized in assays used to diagnose malaria, as well as to determine whether Plasmodium vivax is the species responsible for the infection.

The U.S. Government has rights in this invention by virtue of Grant Nos.RO1 AI 24710 from The National Institutes of Health andDPE-5979-A-00-0006 from the Agency for International Development.

This is a continuation of application Ser. No. 08/478,417, filed Jun. 7,1995 now abandoned, which is a continuation of application Ser. No.08/072,610, filed Jun. 2, 1993 and issued on Jul. 2, 1996 as U.S. Pat.No. 5,532,133.

SUBJECT AREA OF THE INVENTION

This invention is directed to novel species-specific malarialpolypeptides which are secreted into the plasma of a susceptiblemammalian host after infection, and to antibodies directed against thoseproteins. The polypeptides and/or antibodies are utilized in assays usedto diagnose malaria, as well as to determine whether Plasmodium vivax isthe species responsible for the infection.

BACKGROUND OF THE INVENTION

Malaria is transmitted by the bite of the Anopheles mosquito. Minutesafter infection, sporozoites (the mosquito-hosted stage of the malarialparasite) enter hepatocytes of the susceptible mammal where theymultiply by schizogony and develop into merozoites. Rupture of theinfected cells releases the merozoites into the blood, where they entererythrocytes to begin a phase of asexual reproduction. During acuteinfections, malarial parasite protein antigens are known to be released,accumulate, and circulate in the plasma of infected individuals (Wilsonet al., The Lancet, Jul. 26, 1969; Wilson et al., International Journalfor Parasitology 3:511-520, 1973; Wilson et al., Parasitology,71:183-192; Wilson, Nature, 284:451-452, 1980). The release of theseantigens of parasitic origin can occur at the time that infectederythrocytes rupture to allow invasive merozoites to invade new redblood cells. The antigens that spill into the host plasma are those thathave accumulated in the host cell cytoplasm and internal membranousstructures.

Additionally, release of antigen can occur during the intraerythrocyticgrowth of the parasite as it matures from the ring stage, the stagewhich invades the erythrocyte, through the trophozoite stage, and intoschizogony when the parasite differentiates into merozoites. Release ofantigens at this time involves transport of the protein from theparasite across the parasitophorous vacuole and its membrane, across thehost cell cytoplasm to the infected erythrocyte membrane, and thensecretion as an intact soluble protein into the plasma of the host. OneP. falciparum protein, PfHRP-2 (Histidine Rich Protein-2) has beendescribed that follows this route of transport and is secreted into theculture supernatant or found in plasma (Wellems et al., Proc. Natl.Acad. Sci. USA, 83:6065-6069, 1986; Howard et al., J. of Cell. Biol.,103:1269-1277, 1986; Rock et al., Parasitology, 95:209-227, 1987; Pantonet al., Mol. and Biochem. Parasitology, 35:149-160, 1989). A search forHRP analogues in P. vivax using PfHRP gene probes and HRP-antisera gaveonly negative results (Rock et al., Parasitology, 95:209-227, 1987; J.Barnwell, unpublished results).

There is a need in the field for antibodies specific for a P. vivaxblood stage protein in a diagnostic assay. The prior art assays based onantibodies specific for blood stage proteins have been specific only forP. falciparum (Khusmith, Southeast Asian J Trop Med Public Health(THAILAND), 19:21-6, 1988) or have involved the use ofpanspecies-specific antibodies, so no existing assays are specific forP. vivax (Gao et al., Southeast Asian J Trop Med Public Health(THAILAND), 22:393-6, 1991 and James, MA et al., American Society ofTropical Medicine and Hygiene, Seattle, Wash., Nov. 16-19, 1992, Abs.#135, pp. 145-146) P. vivax has latent liver stages, termed hypnozoites,which are reactivated and reinitiate blood stage parasitemias.Hypnozoites are eliminated by treatment with primaquine, but are notaffected by chloroquine, which acts only on blood stage parasites. As P.falciparum does not produce hypnozoites, it is important to identifycorrectly the Plasmodium species responsible for infection in order toprovide the appropriate course of chemotherapy for complete cure. Theincreased prevalence of drug resistant strains in certain species alsomakes it important to identify the species involved so correctchemotherapy can be given. Thus, there is a need for a method andreagents adapted for differential diagnosis of P. vivax malaria.

However, a number of criteria should be met by a particular proteinantigen considered as a potential diagnostic target. First, it should besoluble and relatively stable and not rapidly degraded and/or rapidlyremoved from circulation. Second, the antigen should contain epitopesunique to a species to allow specific diagnosis and preferably bewell-conserved within all or most isolates of a species. Additionally,it should be relatively abundant to allow detection at low parasitemia.As discussed below, the proteins of this invention fulfill most or allof these requirements.

SUMMARY OF THE INVENTION

Secreted species-specific blood stage antigens have now been identifiedfrom a major human malaria parasite species, P. vivax. Two particularsuch proteins are designated P. vivax Erythrocyte Secreted Protein-1(PvESP-1) and P. vivax Erythrocyte Secreted Protein-2 (PvESP-2). Theseantigens and fragments thereof have unique P. vivax-specific epitopeswhich permits their use in differential determination of P. vivaxmerozoites. Antibodies can be and have been elicited against uniqueepitopes of such P. vivax proteins and used in assays which not onlydiagnose malaria, but also selectively identify P. vivax as the specieshaving caused the infection.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are Western immunoblots of P. vivax trophozoite infectederythrocytes probed with antibodies specific for PvESP-1 and PvESP-2.They show that mAb 1D11.G10 reacts with a 225 KD protein, while mAb3D4.E2 and 1A3.B4 react with a 70 KD protein.

FIGS. 2A and 2B are Western immunoblots of P. vivax infectederythrocytes and supernatant from cultures which were matured from ringstage to late-staged trophozoites in vitro. The blots are probed withmAbs specific for PvESP-1 (2A) and PvESP-2 (2B). They show that bothPvESP-1 and PvESP-2 are present in isolated infected erythrocytes and inthe culture medium.

FIG. 3A is a schematic representation of the P. vivax ESP-1 gene andstructural features of the deduced protein. FIG. 3B is a partialrestriction map of the P. vivax ESP-2 gene.

FIG. 4A is an immunoblot of P. vivax culture supernatants and plasmafrom P. vivax infected squirrel (Saimiri) monkeys. 4B is an immunoblotof multiple species of Plasmodium in multiple stages probed with PvESP-1specific antibodies. FIGS. 4C and 4D are immunoblots of plasma fromindividuals infected with P. falciparum, P. vivax or both, and alsoprobed with PvESP-1 specific antibodies. This group of figures shows theselective reaction of these antibodies with P. vivax and with proteinsin the plasma of those infected with P. vivax. Similar results can beobtained with PvESP-2 antibodies using immunoblot procedures. (Example5) Similar results for malaria specificity are also obtained for PvESP-1or PvESP-2 antibodies on smears of different species of malariaparasites by indirect immunofluorescence assay.

FIG. 5 is the DNA sequence (SEQ ID NO:1) and deduced amino acid sequence(SEQ ID NO:2) of P. vivax ESP-1 (a sequence listing is providedseparately).

FIGS. 6A and B are the immunoelectron micrographs of P. vivax infectederythrocytes probed with mAb 1D11.G10 and mAb 3D4.A2, respectively.

FIGS. 7A and B are immunofluorescent assays of P. vivax infectederythrocytes reacted with fluorescence-conjugated mAb 1D11.G10 and mAb3D4.A2, respectively.

DETAILED DESCRIPTION OF THE INVENTION

All U.S. patents and references referred to herein are herebyincorporated by reference in their entirety. In case of conflict, thepresent disclosure controls.

The following definitions apply to the terms as used in this applicationonly and should not be construed to necessarily apply to uses of theterms in other art.

"Stringent conditions" are as defined by Southern et al. in J. of Mol.Bio., 98:503 and as detailed in Maniatis, Molecular Cloning: ALaboratory Manual, 2nd ed., Chapter 9, 1989.

"Immunoreactive fragment" means a fragment of an antigen that isrecognized by an antibody raised against the entire antigen.

"Immunoreactive analog" means a polypeptide which differs from anaturally occurring or recombinant protein by the substitution, deletionand/or addition of one or more amino acids but which retains the abilityto be recognized by an antibody raised against the entire protein. Anonlimiting example is a carrier/antigen fusion polypeptide of the wholeantigen or an immunoreactive fragment thereof, where the antigen orfragment can be embedded within the carrier polypeptide or linked to thecarrier polypeptide at either end.

"Detecting" means determining the presence (or absence) or quantity of asubstance (e.g. an antigen-antibody complex).

"Antibody" includes intact antibody molecule or fragments thereof thatrecognize antigen (e.g. Fab or F(ab')2 fragments) and can be ofpolyclonal or monoclonal type.

"Epitope" means any antigenic determinant responsible for immunochemicalbinding with an antibody molecule. Epitopes usually reside withinchemically active surface groupings of protein molecules (includingamino acids and often also sugar side-chains) and have specificthree-dimensional structural characteristics and specific chargecharacteristics.

"Peptide Antigen" means a peptide, dipeptide, or polypeptide that canelicit (or react with) antibodies recognizing a particular protein.

The search for secreted blood stage antigens for P. vivax began bymaking monoclonal antibodies specific for blood stage parasites. Asdescribed in detail in Example 1, the mAbs were made by conventionaltechniques through the fusion of spleen cells isolated from a mouseimmunized with P. vivax infected red blood cells with mouse myelomacells to produce mAb secreting hybridomas. Three of these mAbs werefound to react with the P. vivax proteins described herein. Theseproteins have been shown to be synthesized by the parasite by severalcriteria. First, mAbs do not react with uninfected erythrocytes as shownby control experiments and the specificity of the mAb for P. vivax,described in Example 6. A reaction to all species would be seen if theproteins were erythrocytic. Second, as seen by IFA and IEM, the mAbs donot react with uninfected erythrocytes which are present in thepreparations. (See FIGS. 6 and 7) Third, the mAbs have been used toimmunoprecipitate radiolabelled proteins from extracts of parasites thathave been biosynthetically labelled with ³⁵ S-methionine. These resultsindicate that the mAbs recognize P. vivax proteins.

Specifically, mAb 1D11.G10 recognizes a P. vivax protein ofapproximately 225,000 daltons in size as judged by SDS-PAGE (FIG. 1A,lane 1). The hybridoma which produces mAb 1D11.G10 has been depositedwith the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110 on May 26, 1993 and is Accession No. HB 11365. Theprotein recognized by this mAb has been designated P. vivax. ESP-1 orPvESP-1. It is found in the culture supernatant when intact infectederythrocytes are incubated in vitro for 10-24 hours (FIG. 2A, lanelabelled SUP) as well as supernatant collected from in vitro cultures ofrupturing mature schizont infected red cells (lane labelled IRBC). Thesedata indicate that this protein is secreted. It is localized byinmunofluorescent assay (IFA) and immuno-electron microscopy (IEM) tothe erythrocyte membrane of infected erythrocytes. (FIGS. 6A and 7A)

mAbs 3D4.A2 and 1A3.B4 recognize a P. vivax protein of approximately70,000 daltons in size as judged by SDS-PAGE (FIG. 1B, lane 1). Thehybridomas which produces mAbs 3D4.A2 and 1A3.B4 have been depositedwith the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110 on May 26, 1993 and are Accession Nos. HB 11367 andHB 11366, respectively. The protein recognized by these mAb has beendesignated P. vivax ESP-2 or PvESP-2. Like PvESP-1, PvESP-2 is found inthe supernatants of in vitro cultured intact trophozoite-infectederythrocytes (FIG. 2B, lane labelled SUP), and thus is a secretedprotein. The PvESP-2 protein is also found in culture supernatantscollected after schizont-infected erythrocytes have ruptured andreleased merozoites (lane labelled IRBC). The protein is localized byIFA and IEM to the caveola-vesicle complexes (CVC) of P. vivax infectederythrocytes. (FIGS. 6B and 7B.) The CVC are membranous sac-likevesicles attached to and contiguous with areas of flask-shapedindentations called caveolae in the erythrocyte plasma membrane.

Polyclonal antibodies can also be produced, for example, using isolatedPvESP-1 and/or PvESP-2 or fragments thereof. General methodology formaking such polyclonal antibodies is well-known in the art, and can bemade using protocols similar to that described in Pink et al. (Eur. J.Immunol. 4:426-429, 1974).

The mAbs described above were used to screen a λZAP recombinant phagelibrary of the P. vivax genome, although other equivalent P. vivaxlibraries could have been used. The preparation of this library isdescribed in Example 2. After induction, mAb 1D11.G10 specificallyrecognized one plaque, designated PvMB3.3.1. The 3.34 kB plasmid insertwas isolated and sequenced. The resulting sequence is SEQ ID No: 1. Thenucleic acid sequence was analyzed for open reading frames (ORF) and thededuced amino acid sequence of the encoded protein was determined. Theamino acid sequence is SEQ ID No: 2. A schematic structure of the geneand features of the encoded protein is presented in FIG. 3A. The geneappears to be missing a small portion of its 5' end.

As shown in FIGS. 3A and 5, the deduced amino acid sequence has aninitial (N-terminal) sequence of hydrophobic amino acids. This isfollowed by a short 139 base pair (bp) intron with typical malariaintervening sequence splice sites. There follows a 2964 bp ORF, endingin the TAA stop codon which is 53 bp before the end of the cloned 3.34kB insert DNA. A protein having this deduced peptide sequence ishydrophilic with a low pI (3), consistent with a large proportion ofglutamate (Glu or E) residues in the deduced amino acid sequence.

As indicated in the Figures, there are two sets of repeated amino acidunits in the sequence. One repeat unit is characterized by the sequenceD(L/M)EAGEE(A/T)G residues 129 to 137, 138 to 146, 147 to 155, 156 to164, 165 to 173, 174 to 182 and 183 to 191 of SEQ. ID NO:2. Thissequence (SEQ ID NO:3) is repeated 7 times at the N-terminal end of theprotein. The second repeat is located in the C-terminal portion of theprotein, has the sequence EEVEEVP (SEQ ID NO:4) residues 649 to 655, 659to 665, 669 to 675, 676 to 682, 686 to 692, 693 to 699, 700 to 706, 707to 713, 714 to 722, and 723 to 728 of SEQ ID NO:2, and is repeated 10times. The hydrophobic amino acid sequence could potentially be, asjudged by its computer analyzed hydrophobicity profile, a transmembranedomain, or a leader or signal peptide sequence, or act as both.Completion of the 5' gene sequence will shed more light on thesepossibilities, and is well within the skill of the art in light of thepresent disclosure.

To determine the remainder of the gene sequence, the complete intactgene can be isolated and sequenced using a large DNA fragment, forexample, in a Lambda replacement vector such as Lambda DASH (Stratagene,Lajolla, Calif.) or equivalent library using the insert as a probe.Methodology for this is provided by Galinski et al. (Cell, 69:1213-1226,1992) or other similar methods. Alternatively, the 5' end could beisolated by the PCR amplification or other method of amplification ofthe cDNA using appropriate primers, for example, as described by Frohmanet al. (Proc. Natl. Acad. Sci. USA, 85:8998-9002, 1988).

The MB3.3.1 plasmid expresses in E. coli a large recombinant proteinrecognized by the mAb 11D.G10 in Western immunoblots. The topmost bandrecognized is approximately 205-210 Kd in size, confirming that a smallportion of the complete PvESP-1 gene remains unsequenced since thenative protein migrates in SDS-PAGE at 225 Kd under identicalconditions. This protein is easily isolated from the culture, usingwell-known techniques (Maniatis, Molecular Cloning: A Laboratory Manual,2nd ed., Chapter 18, 1989). Mouse and rabbit antisera generated byimmunization with the 1D11.G10 affinity purified recombinant proteinrecognizes both the recombinant and native PvESP-1 indicating that therecombinant phagmid MB3.3.1 authentically encodes PvESP-1.

Screening of the λZAPII expression libraries with the mAB 3D4.E2revealed one phage plaque recognized by antibody. This clone, PvMB2.5.1,was found to contain a plasmid having a 3.7 kB insert. This plasmid hasbeen deposited with the ATCC, Bethesda, Md. on May 26, 1993 havingAccession No. 69318. A partial restriction map of the plasmid insert isdepicted in FIG. 3B. The sequence of this DNA is easily obtainableusing, e.g., traditional nested deletion and subcloning techniques orusing nucleic acid primers obtained by dideoxy sequencing of the insert(Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Chapter 13,1989).

The MB2.5.1 plasmid expresses a recombinant polypeptide of approximately60 KD, which is easily isolated from the culture medium using standingprotein isolation techniques (Maniatis, Molecular Cloning: A LaboratoryManual, 2nd ed., Chapter 18, 1989). The size of this protein suggeststhat a portion of the coding region of this protein is not present inthe insert, as the native protein migrates under identical conditions inSDS-PAGE at approximately 70 KD. The 60 KD polypeptide is recognized bymAb 3D4.E2 in Western blots, indicating the recognized epitope isencoded by the insert. Additionally confirmation that plasmid MB 2.5.1authentically encodes PvESP-2 can be done as for PVESP-1, by immunizingwith recombinantly expressed antigen and using the antisera to determineif it recognizes native 70 kD PvESP-2.

Both PvESP-1 and PvESP-2 are successfully expressed in E. coli andexpression of these proteins in other systems, such as viral systems,other bacterial systems, yeast systems or mammalian cell culture, isalso contemplated and is well within the skill of the art. Using suchalternate expression systems may be preferred if glycosylation or otherpost-translational modification is desired.

Purified native or recombinant peptide antigens of the present inventioncan be used in immunogenic preparation to raise additional antibodies.Such preparations will include immunogenically effective amounts of thepresent antigens as well as pharmaceutically acceptable vehicles,carriers, buffers, fillers adjuvants and/or diluents.

Peptide antigens of the present invention can be purified by well-knownchromatographic techniques. Examples include SDS or native PAGE gelelution, size exclusion gel filtration, anion/cation exchange, antibodyaffinity, protein binding to immobilized glutathione or MBP fusionpartner binding to immobilized amylase, metal binding with apolyhistadine linker peptide after expression in a suitable plasmidvector and cell host, or combinations thereof.

As is evident to one of ordinary skill, it is only certain portions orepitopes of the proteins which are recognized by Mabs. The portions ofthe protein(s) containing the relevant epitopes can be identified.Fragments of the gene can be subcloned into the appropriate readingframe of a plasmid expression vector and used to transfect E. coli (orother host system) and expressed by induction. Defined gene fragmentscan be generated using restriction enzymes with cutting sites within thegene. Alternatively, appropriate oligonucleotide primers can be used ina PCR-based amplification reaction to engineer the DNA fragment to besubcloned into the plasmid expression vector. Once the expression vectoris constructed, the recombinant immunoreactive fragments (or analogs,e.g. as a fusion polypeptide) are expressed. The produced fragments arethen reacted with antibodies as above in Western immunoblots, ELISAtests, or other immunochemical assay methods to determine which portionor portions of the protein specifically interact with the antibodies.These methods work well for defining relatively large or small regionsof a protein to locate the corresponding epitope and is effective inidentifying conformation-dependent (discontinuous) epitopes, or linearepitopes. An alternative method for identifying antigenic determinantsis the use of overlapping synthetic peptides of 8-15 amino acids thatcorrespond to the deduced amino acid sequence of the gene. Reactivity ofthese peptides can be determined using ELISA-based assays, such as themethodology of Geysen et al. (Journal of Immunological Methods,102:259-274, 1987) or using commercial-based peptide synthesis kits,i.e., Pepscan or Inimotope. (Cambridge Research Biochemicals, Valleystream, N.Y. and Chiron, Emeryville, Calif., respectively) This methodis especially effective in determination of linear epitopes.

The detection of parasite antigens present in a biological fluid (e.g.plasma), such as PvESP-1 and PvESP-2, can constitute a method for thediagnosis of acute or chronic P. vivax malaria infections. To be useful,such an antigen should contain epitopes unique to the P. vivax speciesto allow specific diagnosis and differential diagnosis from othermalarial infections, and should preferably be conserved within all ormost isolates of that species (more than one antigens can be used togenerate antibodies if necessary to accommodate strain variations).Either monoclonal antibodies or polyclonal antibodies could be used inthe assay, with monoclonals preferred. The epitopes recognized by themonoclonal antibodies 1D11.G10 (anti-PvESP-1) and 3D4.E2 (anti-PvESP-2)are present in all or most P. vivax so far tested (25/26 for 1D11.G10and 26/26 for 3D4.E2). However, these antigens are not present in P.falciparum (FIG. 4A, lane 3), P. malariae (lane 2), P. coatneyi (lane4), P. knowlesi (lane 5), or P. berghei (lane 1). 1D11.G10 doescross-react with P. cynomolgi (lane 6), a simian malaria parasite veryclosely related to P. vivax (lane 7), but never found to occur as anaturally acquired human malaria infection. The mAB 3D4.E2 in particularonly recognizes P. vivax and thus far does so 100% of the time. Ofcourse, any strain differences that may be encountered may be accountedfor in an assay by provision of additional appropriate antibodies, or byprovision of antibodies directed to inter-strain conserved epitopes,which can be conveniently raised against recombinant versions of PvESP-1and PvESP-2 as well as immunoreactive fragments and analogs thereof.

The detected antigens are relatively stable in vivo; that is, they arenot rapidly degraded and/or removed from circulation. PvESP-1 andPvESP-2 can be detected by Western immunoblot in the plasma of squirrel(Saimiri) monkeys experimentally infected with P. vivax (FIG. 4A, lane3) and in the plasma of humans from endemic areas that are infected withP. vivax (FIGS. 4C, lanes 8-11 and 4D, lanes 5-7). The antigens are notdetected in plasma of individuals infected only with P. falciparum(FIGS. 4C, lanes 3-7 and 4D, lanes 6 and 7), the major human malariaparasite that must be differentiated from P. vivax. The squirrel monkeymodel closely approximates what would occur in naturally infectedhumans, but under more controlled conditions than that of work conductedin the field within endemic areas. In Saimiri monkey infections, theantigen can be detected with the present antibodies when there are 1000parasites/μl blood. In humans, early acute infections are detected.Again, as is evident to one of ordinary skill, the isolation of thegenes means that high-titer, high-affinity (e.g., of the order of 10¹⁰liters/mol) antibodies can be produced using standard methodology. Theseantibodies will be used to increase the sensitivity and specificity ofthe assay.

Other serological assay formats based on antigen capture and a reportersignal have produced similar results as described above using mABs3D4.1E2 and 1D11.G10. Based on these successes, it is anticipated thatthese mAbs or others to be produced using the recombinant proteins orimmunogenic fragments thereof can be adapted for use in immunoassaysystems (using either labelled Abs or labelled antigens) well-known inthe diagnostic testing art.

All well-known methods of labelling antibodies are contemplated,including without limitation enzymatic conjugates, direct labelling withdye, radioisotopes, fluorescence, or particulate labels, such asliposome, latex, polystyrene, and colloid metals or nonmetals. Multipleantibody assay systems, such as antigen capture sandwich assays, arealso within the scope of this invention. Further, competitiveimmunoassays involving labelled protein or assays using the labelledprotein to detect serum antibodies are also contemplated forms of thediagnostic assays of the present invention. Beyond diagnostic assayswhich occur in solution, assays which involve immobilized antibody orprotein are also considered within the scope of the invention. (See, forexample, Miles et al., Lancet 2:492, 1968; Berry et al., J. Virol. Met.34:91-100, 1991; Engvall et al., G. Immunochemistry, 8:871, 1971, Tom,Liposomes and Immunology, Elsevier/North Holland, New York, N.Y., 1980;Gribnau et al., J. of Chromatogr. 376:175-89, 1986 and all referencescited therein).

Examples of the types of labels which can be used in the presentinvention include, but are not limited to, enzymes, radioisotopes,fluorescent compounds, chemiluminescent compounds, bioluminescentcompounds, particulates, and metal chelates. Those of ordinary skill inthe art will know of other suitable labels for binding to the monoclonalor polyclonal antibody (or to an antigen) or will be able to ascertainthe same by the use of outine experimentation. Furthermore, the bindingof these labels to the monoclonal or polyclonal antibody (or antigen)can be accomplished using standard techniques commonly known to those ofordinary skill in the art.

One of the ways in which an assay reagent (generally, a monoclonalantibody, polyclonal antibody or antigen) of the present invention canbe detectably labeled is by linking the monoclonal antibody, polyclonalantibody, or antigen to an enzyme. This enzyme, in turn, when laterexposed to its substrate, will react with the substrate in such a manneras to produce a chemical moiety which can be detected as, for example,by spectrophotometric or fluorometric means.

Examples of enzymes which can be used to detectably label the reagentsof the present invention include malate dehydrogenase, staphylococcalnuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholineesterase.

The presence of the detectably labeled reagent of the present inventioncan also be detected by labeling the reagent with a radioactive isotopewhich can then be determined by such means as the use of a gamma counteror a scintillation counter. Isotopes which are particularly useful forthe purpose of the present invention are ³ H, ¹²⁵ I, ³² P, ³⁵ S, ¹⁴ C,⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe and ⁷⁵ Se.

It is also possible to detect the binding of the detectably labeledreagent of the present invention by labeling the monoclonal orpolyclonal antibody with a fluorescent compound. When the fluoroescentlylabeled reagent is exposed to light of the proper wave length, itspresence can then be detected due to the fluorescence of the dye. Amongthe most commonly used fluorescent labelling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine.

The reagents according to the invention also can be detectably labeledusing fluorescent emitting metals such as ¹⁵² Eu, or others of thelanthanide series. These metals can be attached to the reagent moleculeusing such metal chelating groups as diethylenetriaminepentaacetic acid(DTPA) or ethylenediaminetetraacetic acid (EDTA) and salts thereof.

The reagents of the present invention also can be detectably labeled bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged reagent is then determined by detecting thepresence of luminescence that arises during the course of the chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the reagent ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent reagent is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Another technique which may also result in greater sensitivity when usedin conjunction with the present invention consists of coupling themonoclonal or polyclonal antibody of the present invention to lowmolecular weight haptens. The haptens can then be specifically detectedby means of a second reaction. For example, it is common to use suchhaptens as biotin (reacting with avidin) or dinitrophenol, pyridoxal andfluorescamine (reacting with specific antihapten antibodies) in thismanner.

Any biological sample containing the detectable yet unknown amount of P.vivax specific blood-stage antigen can be used to assay. Normally, thesample is preferably a liquid, such as, for example, urine, saliva,cerebrospinal fluid, blood, serum and the like, or a solid orsemi-solid, such as, for example, tissue, feces and the like.

It appears that 1D11.G10 may recognize a repeated epitope since it hasbeen successfully used in a two-site antigen capture immunoassay usingthe same mAb for capture and an alkaline phosphatase labelled mAb or mAbconjugated to liposomes encapsulating a marker dye for the reporterantibody (Example 6). Eleven of 15 plasma samples from P. vivax infectedindividuals were positive by alkaline phosphatase conjugated antibody.Thirteen of 15 samples were positive by liposome conjugated antibody.None of 18 P. falciparum infected plasma samples were positive.Therefore, antibodies to PvESP-1 and PvESP-2 appear quite effective whenused in a diagnostic assay of P. vivax infection and further, suchassays appear to specifically identify P. vivax infection. It isanticipated that assays based on mAb specific for particular epitopesand selected for their high titer and/or affinity will serve to increasethe specificity and sensitivity of the assay.

In general, increases in sensitivity are a development consideration andare achieved by optimization of all reagents, including theconcentrations conjugated to reporter systems, adsorbed to solid phasesurfaces, specificity of the Abs, and affinity of the Abs. These stepsare routinely done and evaluated during assay development and are wellwithin the skill of those working in the art.

As is evident to one of ordinary skill, the diagnostic assay of thepresent invention includes kit forms of such an assay. This kit wouldinclude anti-PvESP-1 and/or anti-PvESP-2 monoclonal or polyclonalantibodies (raised against whole PvESP or immunoreactive fragments oranalogs thereof) which can be optionally immobilized, as well as anynecessary reagents and equipment to prepare the biological sample forand to conduct analysis, e.g. preservatives, reaction media such asnontoxic buffers, microtiter plates, micropipettes, etc. The reagent(Abs and/or antigens) can be lyophilized or cryopreserved. As describedabove, depending on the assay format, the antibodies can be labelled, orthe kit can further comprise labelled PvESP-1 or PvESP-2 protein orfragments or analogs thereof containing the relevant epitopes.

The types of immunoassays which can be incorporated in kit form aremany. Typical examples of some of the immunoassays which can utilize theantibodies of the invention are radioimmunoassays (RIA) andimmunometric, or sandwich, immunoassays.

"Immunometric assay" or "sandwich immunoassay", includes simultaneoussandwich, forward sandwich and reverse sandwich immunoassays. Theseterms are well understood by those skilled in the art. Those of skillwill also appreciate that the monoclonal antibodies, polyclonalantibodies and/or antigens of the present invention will be useful inother variations and forms of immunoassays which are presently known orwhich may be developed in the future. These are intended to be includedwithin the scope of the present invention.

In a forward sandwich immunoassay, a sample is first incubated with asolid phase immunoadsorbent containing monoclonal or polyclonalantibody(ies) against the antigen. Incubation is continued for a periodof time sufficient to allow the antigen in the sample to bind to theimmobilized antibody in the solid phase. After the first incubation, thesolid phase immunoadsorbent is separated from the incubation mixture andwashed to remove excess antigen and other interfering substances, suchas non-specific binding proteins, which also may be present in thesample. Solid phase immunoadsorbent containing antigen bound to theimmobilized antibody is subsequently incubated for a second time withsoluble labeled antibody or antibodies. After the second incubation,another wash is performed to remove unbound labeled antibody(ies) fromthe solid phase immunoadsorbent and removing non-specifically boundlabeled antibody(ies). Labeled antibody(ies) bound to the solid phaseimmunoadsorbent is then detected and the amount of labeled antibodydetected serves as a direct measure of the amount of antigen present inthe original sample.

Alternatively, labeled antibody which is not associated with theimmunoadsorbent complex can also be detected, in which case the measureis in inverse proportion to the amount of antigen present in the sample.Forward sandwich assays are described, for example, in U.S. Pat. Nos.3,867,517; 4,012,294 and 4,376,110.

In carrying out forward immunometric assays, the process may comprise,in more detail: (a) first forming a mixture of the sample with the solidphase bound antibody(ies) and incubating the mixture for a time andunder conditions sufficient to allow antigen in the sample to bind tothe solid phase bound antibody(ies), (b) adding to the mixture aftersaid incubation of step (a) the detectably labeled antibody orantibodies and incubating the new resulting mixture for a time and underconditions sufficient to allow the labeled antibody to bind to theantigen-antibody complex on the solid phase immunoadsorbent; (c)separating the solid phase immunoadsorbent from the mixture after theincubation in step (b); and (d) detecting either the labeled antibody orantibodies bound to the antigen-antibody complex on the solid phaseimmunoadsorbent or detecting the antibody not associated therewith.

In a reverse sandwich assay, the sample is initially incubated withlabeled antibody(ies), after which the solid phase immunoadsorbentcontaining multiple immobilized antibodies is added thereto, and asecond incubation is carried out. The initial washing step of a forwardsandwich assay is not required, although a wash is performed after thesecond incubation. Reverse sandwich assays have been described, forexample, in U.S. Pat. Nos. 4,098,876 and 4,376,110.

In carrying out reverse immunometric assays, the process may comprise,in more detail; (a) first forming a mixture of the sample with thesoluble detectably labeled antibody for a time and under conditionssufficient to allow antigen in the sample to bind to the labeledantibody; (b) adding to the mixture after the incubation of step (a) thesolid phase bound antibodies and incubating the new resulting mixturefor a time and under conditions sufficient to allow antigen bound to thelabeled antibody to bind to the solid phase antibodies; (c) separatingthe solid phase immunoadsorbent from the incubating mixture after theincubation in step (b); and (d) detecting either the labeled antibodybound to the solid phase immunoadsorbent or detecting the labeledantibody not associated therewith.

In a simultaneous sandwich assay, the sample, the immunoadsorbent havingmultiple immobilized antibodies thereon and labeled soluble antibody orantibodies are incubated simultaneously in one incubation step. Thesimultaneous assay requires only a single incubation and does notinclude washing steps. The use of a simultaneous assay is by far thepreferred one. This type of assay brings about ease of handling,homogeneity, reproducibility, and linearity of the assays and highprecision. The sample containing antigen, solid phase immunoadsorbentwith immobilized antibodies and labeled soluble antibody or antibodiesis incubated under conditions and for a period of time sufficient toallow antigen to bind to the immobilized antibodies and to the solubleantibody(ies). In general, it is desirable to provide incubationconditions sufficient to bind as much antigen as possible, since thismaximizes the binding of labeled antibody to the solid phase, therebyincreasing the signal. Typical conditions of time and temperature aretwo hours at 45° C., or twelve hours at 37° C. Antigen typically bindsto labeled antibody more rapidly than to immobilized antibody, since theformer is in solution whereas the latter is bound to the solid phasesupport. Because of this, labeled antibody may be employed in a lowerconcentration than immobilized antibody, and it is also preferable toemploy a high specific activity for labeled antibody. For example,labeled antibody might be employed at a concentration of about 1-50 ngper assay, whereas immobilized antibody might have a concentration of10-500 ng per assay per antibody. The labeled antibody might have aspecific activity with, for instance, one radioiodine per molecule, oras high as two or more radioiodines per molecule of antibody.

Of course, the specific concentrations of labeled and immobilizedantibodies, the temperature and time of incubation as well as otherassay conditions can be varied, depending on various factors includingthe concentration of antigen in the sample, the nature of the sample andthe like. Those skilled in the art will be able to determine operativeand optimal assay conditions for each determination by employing routineexperimentation.

After the single incubation period, the solid phase immunoadsorbent isremoved from the incubation mixture. This can be accomplished by any ofthe known separation techniques, such as sedimentation andcentrifugation. A washing step is not required prior to detection ofbound labeled antibody. Detection can be performed by a scintillationcounter, for example, if the label is a radioactive gamma-emitter, or bya fluorometer, for example, if the label is a fluorescent material. Inthe case of an enzyme label, the detection can be done by calorimetricmethods employing a substrate for the enzyme.

In carrying out the simultaneous immunometric assay on a samplecontaining a multivalent antigen, the process may comprise, in moredetail:

(a) simultaneously forming a mixture comprising the sample, togetherwith the solid phase bound antibody and the soluble labeled antibody orantibodies;

(b) incubating the mixture formed in step (a) for a time and underconditions sufficient to allow antigen in the sample to bind to bothimmobilized and labeled antibodies;

(c) separating the solid phase immunoadsorbent from the incubationmixture after the incubation; and

(d) detecting either labeled antibody bound to the solid phaseimmunoadsorbent or detecting labeled antibody not associated therewith.

Other such steps as washing, stirring, shaking filtering and the likemay of course be added to the assays, as is the custom or necessity forany particular situation.

In the preferred mode for preforming the assays it is important thatcertain "blockers" be present in the incubation medium (usually addedwith the labeled soluble antibody). The "blockers" are added to assurethat non-specific proteins, protease, or human antibodies to mouseimmunoglobulins present in the experimental sample do not cross-link ordestroy the monoclonal or polyclonal antibodies on the solid phasesupport, or the radiolabeled indicator antibody, to yield false positiveor false negative results. The selection of "blockers" therefore addssubstantially to the specificity of the assays described in the presentinvention.

It has been found that a number of nonrelevant (i.e., nonspecific)monoclonal or polyclonal antibodies of the same class or subclass(isotype) as those used in the assays (e.g., IgG1, IgG 2a2, IgM, etc.)can be used as "blockers". The concentration of the "blockers" (normally1-100, μg/μl) is important, in order to maintain the proper sensitivityyet inhibit any unwanted interference by mutually occurring crossreactive proteins in human serum. In addition, the buffer systemcontaining the "blockers" needs to be optimized. Preferred buffers arethose based on weak organic acids, such as imidazole, HEPPS, MOPS, TES,ADA, ACES, HEPES, PIPES, TRIS, and the like, at physiological pH ranges.Somewhat less preferred buffers are inorganic buffers such as phosphate,borate or carbonate. Finally, known protease inhibitors should be added(normally at 0.01-10 microns/ml) to the buffer which contains the"blockers".

There are many solid phase immunoadsorbents which have been employed andwhich can be used in the present invention. Well-known immunoadsorbentsinclude nitrocellulose, glass, polystyrene, polypropylene, dextran,nylon and other materials; tubes, beads, and microtiter plates formedfrom or coated with such materials, and the like. The immobilizedantibodies can be either covalently or physically bound to the solidphase immunoadsorbent, by techniques such as covalent bonding via anamide or ester linkage, or by absorption. Those skilled in the art willknow many other suitable solid phase immunoadsorbents and methods forimmobilizing antibodies thereon, or will be able to ascertain such,using no more than routine experimentation.

Details of the operation and practice of the present invention are setforth in the specific examples which follow. However, these examples arenot to be interpreted as limiting the scope of the present invention.

EXAMPLE 1 Method of Making the Monoclonal Antibodies Specific forPvESP-1 and PvESP-2

Balb/c mice were immunized intraperitoneally with 5×10⁸ purified P.vivax (of the Belem strain infected red blood cells (IRBC) in completeFreud's adjuvant. Immunization was repeated at 2 and 7 weeks usingincomplete Freund's adjuvant and finally at 14 weeks without adjuvant. 3days later, spleen cells from the immunized mouse were fused withmyeloma cell line NY-FOX (Hyclone, Utah; Taggart, Science,219:1228-1230, 1983) according to the basic method of Galfre et al.(Nature, 266:550-552, 1977). Cells were plated directly into microtiterwells and cultured (Rener et al., Proc. Natl. Acad. Sci. USA,77:6797-6799, 1980) such that 1 to 2 weeks later, 1 or more hybridcolonies were observed in all wells. Culture supernatants were collectedand screened by immunofluorescence assay using smears of P. vivaxinfected blood that also contained normal red blood cells. Those cellsproducing antibodies which selectively reacted with IRBCs were expandedand cryopreserved. Secondary screening was performed by SDS-PAGE withhybridoma culture supernatants from expanded cultures that had beenobtained by centrifugation. Those mAbs which reacted with P. vivax bloodstage extracts and culture supernatants (prepared essentiailly asdescribed in Galinski et al., Cell, 69:1213-1226, 1992) were selectedfor further study. Three such mAbs are designated 1D11.G10, 3D4.A2, and1A3.B4.

EXAMPLE 2 Screening of P. vivax λZAPII Expression Library with the mAbs

P. vivax genomic DNA was isolated and digested with mung bean nuclease(U.S. Biochemical) following the procedures of Vernick et al. (Nucl.Acids Res., 16:6883-6896, 1988) and as modified by Galinski et al.(supra) Specifically, the DNA was digested with 42.5-45% formamide. Thedigested DNA was ligated into the λZAPII vector (Stratagene, LaJolla,Calif.) and the resulting phage were used to infect E. coli. Expressionwas induced by growth on IPTG (isopropylthio-β-D-galactoside) containingnitrocellulose plates overlaying the agar plates, and the resultingplaques were screened with the mAbs using standard immuno-detectionmethods (see, for example, Maniatis, Molecular Cloning: A LaboratoryManual, 2nd ed., Chapter 12, 1989).

After screening approximately 3×10⁵ recombinant plaques, mAb 1D11.G10specifically recognized one recombinant phage plaque. This phage clone,PvMB3.3.1 was purified, and in vivo excised with the aid of helper phageR408 (Stratagene, LaJolla, Calif.) to yield the clone as a pBluescriptplasmid retaining the recombinant DNA as a 3.34 kb insert (Short et al.,Nucleic Acids Res., 16:7583, 1988).

After screening approximately 4×10⁵ plaques, mAb 3D4.E2 also revealedone phage plaque recognized by the antibody. This clone, PvMB2.5.1 wasplaque purified and in vivo excised, as above, to yield a pBluescriptplasmid containing the 3.7 kb DNA insert.

EXAMPLE 3 Expression of the Cloned Proteins in E. coli

The isolated pBluescript plasmids where transformed into E. coli andexpression was induced by growth in the presence of IPTG using standardmethodology (Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed.,Chapter 1, 1989). Proteins produced by the cultures were isolated,separated on a gel, blotted and probed using standard techniques(Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Chapter 18,1989). Probing the blot of PvMB3.3.1 with 1D11.G10 revealed multiplebands, the largest of which was 205-210 kD. Probing the blot ofPvMB2.5.1 revealed a 60 kD band. These results indicate that the clonedinserts encode the epitopes recognized by these mAbs.

EXAMPLE 4 Sequencing of the Pv3.3.1 Insert

The insert was directly sequenced from the pBluescript excision plasmid.Nested deletions of 100-300 bp intervals were created using exonucleaseIII and mung bean nuclease (U.S. Biochemical, Cleveland, Ohio) andstandard methodology. DNA sequences were generated using the dideoxytermination method sequencing methodology (Sanger et al., Proc. Natl.Acad. Sci. USA, 74:5463-5467, 1977). The entire PvESP-1 gene wassequenced on both DNA strands, and is SEQ ID No: 1. The deduced proteinsequence (SEQ ID No: 2) was analyzed using Pustell and MacVectorsoftware programs (IBI). GenBank (release 70) and the Swiss Protein DataBank (release 20) were screened for DNA and protein sequence homologiesusing the GCG Sequence Analysis Software Package, Version 7.0 (GeneticsComputer Group, Inc.).

EXAMPLE 5 Cross-reactivity Test with Other Plasmodium Species

FIG. 4A was produced as follows. P. vivax trophozoite-infectederythrocytes (2×10⁴), 25 μl of supernatants from P. vivax trophozoiteand rupturing schizont-infected red blood cell cultures, and 20 μl of a1:10 dilution of P. vivax infected Saimiri monkey plasma were mixed withsample buffer and electro-phoresed on an SDS-PAGE gel. The gel waselectrophoretically transferred to 0.2 μm nitrocellulose (NC) by Westernblot (Towbin, H. et al., Proc. Natl. Acad. Sci. USA, 76:4350, 1979). TheNC was blocked with 3% non-fat dry milk and probed with mAb 1D11.G10 at2 mg/ml in TBS. The blot was washed with TBS/0.05. tween 20 and reprobedwith alkaline phosphatase conjugated anti-mouse IgG (Promega, Madison,Wis.) and developed with P-nitroblue tetrazoliumchloride/5-bromo-4-chloro-3 indolyl phosphate (U.S. Biochemicals,Cleveland, Ohio).

FIG. 4B. was produced as follows. P. vivax IRBC were acquired frominfected Saimiri monkey, P. cynomolgi (M strain) IRBC was from infectedRhesus monkeys, P. knowlesi IRBC were Saimiri monkey, P. coatneyi IRBCwere from Rhesus monkeys, P. falciparum from human rbc in in vitroculture, P. malariae from infected Aotus monkeys, and P. berghei frominfected rats. SDS-PAGE and nitrocellulose transfer were done as abovewith 1×10⁵ parasites/lane dissolved in SDS-PAGE sample buffer. Indirectimmunofluorescence assay was performed by making smears of IRBC onslides and reacting 1D11.G10 or 3D4.E2 with smears and using FITCconjugated goat anti-mouse IgG as secondary antibody with the sameresults as Western blot.

These results show that there is no cross-reactivity with other malarialspecies.

EXAMPLE 6 Diagnostic Assay Using Alkaline Phosphatase and LiposomeConjugated mAb

Unlabelled mAb is absorbed to nitrocellulose sheets (5 μm average poresize) at 5 mg Ab/ml in PBS. The sheet is washed and blocked with 3%non-fat dry milk in TBS. The sheet is layered on an ELISA apparatus(Pierce) and a 96-well plexiglass top (like a slot blot apparatus) issecured in place over the nitrocellulose sheet. Diluted plasma (1:10-100μl) samples are applied to the wells and drawn through thenitrocellulose by vacuum. The wells are washed by vacuum and mAb1D11.G10 conjugated to alkaline phosphatase is applied to the wells.Alkaline phosphatase conjugation was accomplished by the glutaraldehydemethod of Avrameas (Immunochemistry, 6:43, 1967). The alkalinephosphatase conjugated mAb is pulled through the nitrocellulose byvacuum, the wells are washed, and then the developer substrates NBT-BCIPare added to and pulled through the wells. Positive reactions areassessed by the appearance of a purple-violet to blue-black precipitateforming in the wells at the surface of the nitrocellulose. Eleven of the15 plasma samples from P. vivax individuals were positive using thealkaline phosphatase conjugated mAb 1D11.G10. All samples were assessedfor infection with P. vivax, P. falciparum, or both, by Giemsa-stainedthick films of blood samples. False positives thus, would show apositive reaction, but would be negative for P. vivax parasites in thickfilms. No such reactions were seen.

The liposome-based test was similar to the alkaline phosphatase-basedtest. As in the alkaline phosphatase assay, the secondary (reporter) mAbwas conjugated to liposomes that contained a bright red to maroon dye.Thus, the appearance of red on the nitrocellulose was the reportersystem and an enzymatic development step is not needed as in thealkaline phosphatase system. Thirteen of 15 infected samples werepositive using the liposome conjugated 1D11.G10. This assay can also beadapted to a strip test where a mAb or polyclonal Ab is absorbed to a NCstrip that overlays an absorbent pad. Then, test plasma, antibodyconjugated liposomes, and washing solutions are wicked upwards bydiffusion and a positive test is indicated by a red-to-magenta lineacross the NC strip assay.

EXAMPLE 7 Competitive Diagnostic Test for Malaria Which IndicatesSpecific Infection with P. vivax

In a calorimetric immunoassay for PvESP-1 and/or PVESP-2, large,unilamellar phospholipid vesicles approximately 0.2 micrometers indiameter are loaded with high concentrations of Sulforhodamine B or asimilar dye. The PvESP-1 and/or PvESP-2 is coupled tophosphatidylethanolamine or another component of the lipid vesicle, andincorporated into the lipid formulation, thus conferring immunologicalspecificity. Methods of formation of the vesicles, loading the vesicles,and coupling the protein to the phosphatidylethanolamine are disclosedin O'Connell et al. (Clin. Chem, 31:1424-1426). The liposomes are thenused as tracers in simple competitive-binding immunoassays withantibody-coated tubes. The results are read spectrophotometrically.Specific immunoassay methods are described in O'Connell et al., supra,as well as O'Connell, MG and DI, December, 1985, pp. 31-36. As this is acompetitive assay, the less signal seen, the more PvESP-1 and/or PvESP-2will be present in the sample. It is anticipated that this assay will beselective for P. vivax infection, given the selectivity of theantibodies 1D11.G10, 3D4.A2, and 1A3.B4 as shown in Example 5.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3337 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Plasmodium vivax    (vii) IMMEDIATE SOURCE:    (B) CLONE: PvMB3.3.1    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GAATTCCGGTAAAGTAACAACTATGGTTTCGTATCTATATATAACCTTACTAATTTTATC60    TTTTGCTTTTCTTTTAATTCATGCTTCAACAGTAAGATAAAAATAATCTATAAAAACTGC120    TATATATACATATATATTCATAAGTGGCATTTGTGAATTGCGATCATTTAAATTTACGTA180    AAAACAATATTGAAAAAAATTTTTTTTTTTTTTTTTTTTTTGTTCTACAGAACGATTTAG240    AATTGGAAAATGCTTCTGATGATGTTGTAGAGGTGGAGGATCCTTCAAACGACGGTTTAG300    AATTAGAAGAGGAAAATTTTGATGAGAATTCAGGTGATGATGAAACTCTTTTAGATGCTA360    CCCCCGAAGATGACTTTGCCTTAACAGATTTGCCAATTGAAGACGATGAGGAAGTCAACG420    AAACGTTAGATGGAGGTGAATCATTAGGAGAGGTTTCCACTGAAGATATGGAAACAGAAG480    ATGGCTCAACAGATGATACGGAAACAGAAGAAGGACTACCTGGTGATATGGAAGGAGAAG540    AAGAAGCTGGCGATATGGAAGCAGGGGAAGAAGCTGGTGATTTGGAAGCAGGGGAAGAAA600    CTGGCGATTTGGAAGCAGGGGAAGAAACTGGCGATTTGGAAGCAGGGGAAGAAGCTGGTG660    ATTTGGAAGCAGGGGAAGAAACTGGCGATTTGGAAGCAGGGGAAGAAACTGGAGATGCGG720    AAACTGAAGAAGGAGCAACTGGAGATGCGGAAACTGAAAATGGAGCAACTGTGTATGTAG780    ACACAGAAGATAGTTCAGCTGATGGAGCAGAAAAAGTACATGTTCCTGCTCAAGAAAATG840    TACAACCTGCCGATAGTAATGATGCCCTCTTTGGAAGTATTTTGGATAAAGATATAATTT900    TTGATCATATTAAAGATTTCGAGCCACTATTCGAACAAATTGTGGCGGGTACTGCTAAAC960    ATGTTACGGGACAAGAATTGCCAATGAAACCTGTACCATTACCAGTGGCAGAAGAGCCCG1020    CGCAAGTACCAGCGGAAGAATTAGATGCCACTCCAGAGGATGACTTCGCATTAGATGTTA1080    CAGAATCTCCCGAGGAAGTAGAATTAGTATTAGATGAAGAGGCAACTGAAGAAGAATCAA1140    CGGAAGTGGGACCAACGGAAGAAGGACCAACCGAAGAATTAGATGCCACTCCAGAGGATG1200    GATTTCGCATTAGACGAAACTGCAGAAGGAGAAACAGAAGAAACGTAGAGGGAGAAGAAA1260    CAGAAGAAGCTGCAGAAGGAGAAGTATCAGAAGAAACTCCAGAAGGAGAAGAAGAGTTAG1320    AGGCAACTCCAGAGGATGATTTCGCATTAGATGGAACTACATTAGAAGAAACCGAAGAAA1380    CTGCAGAAGGAGAAGAAACCGTAGAGGGAGAAGAAACCGTAGAGGGAGAAGAAACCGTAG1440    AGGGAGAAGAAGCTGCAGAAGGAGAAGAAGAGTTAGAGGCAACTCCAGAGGATGACTTCC1500    AATTAGAAGAACCATCAGGAGAAGGAGAAGGGGAAGGAGAAGGAGAAGGGGAAGGAGAAG1560    GAGAAGCGTTAGTAGCAGTGCCAGTAGTGGCCGAACCGGTAGAAGTAGTGACTCCTGCTC1620    AGCCTGTCAAACCAATGGTCGCTCCAACGGCAGATGAAACTTTATTCGTTGATATCTTAG1680    ATAACGATTTAACGTATGCAGACATTACATCCTTTGAGCCATTATTTAAACAAATCCTCA1740    AGGATCCTGATGCAGGAGAGGCTGTAACAGTACCATCAAAGGAAGCACCTGTACAAGTAC1800    CAGTGGCAGTAGGGCCCGCGCAAGAAGTGCCAACGGAAGAATTGATGCAACTCCAAGAGG1860    ACGATTTCGAATTAGAAGGAACTGCAGAAGCTCCAGAGGAAGGAGAATTAGTATTAGAAG1920    GAGAAGGAGAACCAACGGAAGAAGAGCCAAGAGAAGGAGAGCCAACAGAAGGAGAAGTGC1980    CAGAAGAAGAATTAGAGGCAACTCCAGAGGACGATTTCGAATTAGAAGAACCAACAGGAG2040    AAGAAGTAGAAGAAACCGTAGAGGGCGAAGAAACTGCAGAAGGAGAAGAAGTGGAAGAGG2100    TACCTGCAGAAGTAGAAGAAGTGGAAGAGGTACCTGCAGAAGTAGAAGAAGTGGAAGAGG2160    TACCAGAAGAAGTAGAAGAGGTACCCGCAGAAGTAGAAGAAGTGGAAGAGGTACCAGAAG2220    AAGTGGAAGAGGTACCAGAAGAAGTGGAAGAGGTACCAGAAGAAGTGGAAGAGGTACCAG2280    AAGAAGTGGAAGAAGTGGAAGAAGTAGAAGAAGTAGAGGTACCAGCGGTAGTAGAAGTAG2340    AAGTACCAGCGGTAGTAGAAGAAGAGGTGCCAGAAGAAGTAGAAGAAGAAGAAGAAGAGG2400    AAGAACCAGTAGAGGAAGAAGATGTATTACAATTAGTAATACCATCGGAAGAAGATATAC2460    AATTAGACAAACCAAAGAAAGACGAATTAGGCTCTGGAATTTTATCTATCATCGACATGC2520    ACTACCAAGACGTTCCAAAGGAATTTATGGAAGAAGAAGAAGAAACTGCAGTGTATCCAT2580    TGAAACCAGAAGATTTTGCAAAGGAAGATTCACAATCTACAGAATGGCTCACATTCATTC2640    AAGGCCTAGAAGGCGACTGGGAACGATTAGAAGTGAGCTTAAATAAGGCTAGAGAAAGAT2700    GGATGGAACAAAGAAATAAAGAATGGGCTGGCTGGCTTCGCTTAATTGAAAATAAATGGT2760    CAGAATATAGTCAAATTTCAACAAAAGGAAAGGACCCAGCTGGTTTGAGAAAACGAGAGT2820    GGAGCGACGAGAAATGGAAAAAATGGTTTAAAGCAGAAGTCAAATCCCAAATTGATTCAC2880    ACTTGAAAAAATGGATGAACGACACTCATTCCAATTTATTTAAAATTCTTGTGAAAGATA2940    TGTCACAATTTGAAAACAAGAAAACCAAAGAATGGTTAATGAATCACTGGAAAAAGAACG3000    AACGGGGTTATGGTTCTGAATCATTTGAAGTTATGACCACATCAAAATTATTAAATGTGG3060    CTAAGAGTCGAGAATGGTACCGTGCCAATCCTAATATAAATAGAGAAAGAAGAGAACTCA3120    TGAAATGGTTTCTCCTAAAAGAAAACGAATATTTAGGACAAAGAATGGAAAAAATGGACT3180    CATTGGAAAAAAGTTAAATTTTTTGTGTTCAATTCAATGTGTACAACATTTTCTGGAAAA3240    CGCCTAACCAAGGAAGAATGGAATCAATTTGTTAATGAAATAAAAGTTTGAATTATAGAA3300    AAAAGAACAGATTATTCTCTTATAAAATAAATAATTC3337    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1018 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: YES    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: C-terminal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Plasmodium vivax    (vii) IMMEDIATE SOURCE:    (B) CLONE: PvMB3.3.1    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AsnSerGlyLysValThrThrMetValSerTyrLeuTyrIleThrLeu    151015    LeuIleLeuSerPheAlaPheLeuLeuIleHisAlaSerThrAsnAsp    202530    LeuGluLeuGluAsnAlaSerAspAspValValGluValGluAspPro    354045    SerAsnAspGlyLeuGluLeuGluGluGluAsnPheAspGluAsnSer    505560    GlyAspAspGluThrLeuLeuAspAlaThrProGluAspAspPheAla    65707580    LeuThrAspLeuProIleGluAspAspGluGluValAsnGluThrLeu    859095    AspGlyGlyGluSerLeuGlyGluValSerThrGluAspMetGluThr    100105110    GluAspGlySerThrAspAspThrGluThrGluGluGlyLeuProGly    115120125    AspMetGluGlyGluGluGluAlaGlyAspMetGluAlaGlyGluGlu    130135140    AlaGlyAspLeuGluAlaGlyGluGluThrGlyAspLeuGluAlaGly    145150155160    GluGluThrGlyAspLeuGluAlaGlyGluGluAlaGlyAspLeuGlu    165170175    AlaGlyGluGluThrGlyAspLeuGluAlaGlyGluGluThrGlyAsp    180185190    AlaGluThrGluGluGlyAlaThrGlyAspAlaGluThrGluAsnGly    195200205    AlaThrValTyrValAspThrGluAspSerSerAlaAspGlyAlaGlu    210215220    LysValHisValProAlaGlnGluAsnValGlnProAlaAspSerAsn    225230235240    AspAlaLeuPheGlySerIleLeuAspLysAspIleIlePheAspHis    245250255    IleLysAspPheGluProLeuPheGluGlnIleValAlaGlyThrAla    260265270    LysHisValThrGlyGlnGluLeuProMetLysProValProLeuPro    275280285    ValAlaGluGluProAlaGlnValProAlaGluGluLeuAspAlaThr    290295300    ProGluAspAspPheAlaLeuAspValThrGluSerProGluGluVal    305310315320    GluLeuValLeuAspGluGluAlaThrGluGluGluSerThrGluVal    325330335    GlyProThrGluGluGlyProThrGluGluLeuAspAlaThrProGlu    340345350    AspGlyPheArgIleArgArgAsnCysArgArgArgAsnArgArgAsn    355360365    ValGluGlyGluGluThrGluGluAlaAlaGluGlyGluValSerGlu    370375380    GluThrProGluGlyGluGluGluLeuGluAlaThrProGluAspAsp    385390395400    PheAlaLeuAspGlyThrThrLeuGluGluThrGluGluThrAlaGlu    405410415    GlyGluGluThrValGluGlyGluGluThrValGluGlyGluGluThr    420425430    ValGluGlyGluGluAlaAlaGluGlyGluGluGluLeuGluAlaThr    435440445    ProGluAspAspPheGlnLeuGluGluProSerGlyGluGlyGluGly    450455460    GluGlyGluGlyGluGlyGluGlyGluGlyGluAlaLeuValAlaVal    465470475480    ProValValAlaGluProValGluValValThrProAlaGlnProVal    485490495    LysProMetValAlaProThrAlaAspGluThrLeuPheValAspIle    500505510    LeuAspAsnAspLeuThrTyrAlaAspIleThrSerPheGluProLeu    515520525    PheLysGlnIleLeuLysAspProAspAlaGlyGluAlaValThrVal    530535540    ProSerLysGluAlaProValGlnValProValAlaValGlyProAla    545550555560    GlnGluValProThrGluGluLeuMetGlnLeuGlnGluAspAspPhe    565570575    GluLeuGluGlyThrAlaGluAlaProGluGluGlyGluLeuValLeu    580585590    GluGlyGluGlyGluProThrGluGluGluProArgGluGlyGluPro    595600605    ThrGluGlyGluValProGluGluGluLeuGluAlaThrProGluAsp    610615620    AspPheGluLeuGluGluProThrGlyGluGluValGluGluThrVal    625630635640    GluGlyGluGluThrAlaGluGlyGluGluValGluGluValProAla    645650655    GluValGluGluValGluGluValProAlaGluValGluGluValGlu    660665670    GluValProGluGluValGluGluValProAlaGluValGluGluVal    675680685    GluGluValProGluGluValGluGluValProGluGluValGluGlu    690695700    ValProGluGluValGluGluValProGluGluValGluGluValGlu    705710715720    GluValGluGluValGluValProAlaValValGluValGluValPro    725730735    AlaValValGluGluGluValProGluGluValGluGluGluGluGlu    740745750    GluGluGluProValGluGluGluAspValLeuGlnLeuValIlePro    755760765    SerGluGluAspIleGlnLeuAspLysProLysLysAspGluLeuGly    770775780    SerGlyIleLeuSerIleIleAspMetHisTyrGlnAspValProLys    785790795800    GluPheMetGluGluGluGluGluThrAlaValTyrProLeuLysPro    805810815    GluAspPheAlaLysGluAspSerGlnSerThrGluTrpLeuThrPhe    820825830    IleGlnGlyLeuGluGlyAspTrpGluArgLeuGluValSerLeuAsn    835840845    LysAlaArgGluArgTrpMetGluGlnArgAsnLysGluTrpAlaGly    850855860    TrpLeuArgLeuIleGluAsnLysTrpSerGluTyrSerGlnIleSer    865870875880    ThrLysGlyLysAspProAlaGlyLeuArgLysArgGluTrpSerAsp    885890895    GluLysTrpLysLysTrpPheLysAlaGluValLysSerGlnIleAsp    900905910    SerHisLeuLysLysTrpMetAsnAspThrHisSerAsnLeuPheLys    915920925    IleLeuValLysAspMetSerGlnPheGluAsnLysLysThrLysGlu    930935940    TrpLeuMetAsnHisTrpLysLysAsnGluArgGlyTyrGlySerGlu    945950955960    SerPheGluValMetThrThrSerLysLeuLeuAsnValAlaLysSer    965970975    ArgGluTrpTyrArgAlaAsnProAsnIleAsnArgGluArgArgGlu    980985990    LeuMetLysTrpPheLeuLeuLysGluAsnGluTyrLeuGlyGlnArg    99510001005    MetGluLysMetAspSerLeuGluLysSer    10101015    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: YES    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: C-terminal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Plasmodium vivax    (vii) IMMEDIATE SOURCE:    (B) CLONE: PvMB3.3.1    (ix) FEATURE:    (B) LOCATION: 2    (D) OTHER INFORMATION: Leu or Met    (B) LOCATION: 7    (D) OTHER INFORMATION: Ala or Thr    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    AspXaaGluAlaGlyGluXaaGlu    15    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: YES    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: C-terminal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Plasmodium vivax    (vii) IMMEDIATE SOURCE:    (B) CLONE: PvMB3.3.1    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GluGluValGluGluValPro    15    __________________________________________________________________________

We claim:
 1. An isolated purified peptide antigen comprising an amino acid sequence of PvESP-1, wherein said PvESP-1: (i) is a secreted blood-stage protein from Plasmodium vivax, (ii) is present in detectable amounts in biological samples of individuals infected with Plasmodium vivax, (iii) comprises an epitope not present in other Plasmodium species that cause malaria in humans, and (iv) binds monoclonal antibody 1D11G10;said peptide antigen having the property of eliciting antibodies that recognize said PvESP-1.
 2. An isolated purified peptide antigen comprising an amino acid sequence of PvESP-1 or a fragment thereof, wherein said PvESP-1: (i) is a secreted blood-stage protein from Plasmodium vivax, (ii) is present in detectable amounts in biological samples of individuals infected with Plasmodium vivax and (iii) binds monoclonal antibody 1D11G10;said PvESP-1 or a fragment thereof comprising an epitope not present in other Plasmodium species that cause malaria in humans; said peptide antigen having the property of eliciting antibodies that recognize said PvESP-1.
 3. An isolated purified polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or immunogenic P. vivax fragments thereof not present in other Plasmodium species that cause malaria in humans. 